Disulfide Bonds in Protein Folding and Stability

Many proteins rely on disulfide bonds for the stability of their folded state. This implies that disulfide bond formation can be coupled to folding and assembly, and it is a unique tool in studying both phenomena (, , , , ). An important aspect of disulfide bonds is that their formation and breakage is an oxidation-reduction process, requiring the presence of electron acceptors and donors, respectively. Consequently, disulfide bonds are not made and broken in isolation, but only under certain conditions that are under experimental control. The rates and equilibrium of the process can be varied and measured, making it possible to measure experimentally the relative free energies of individual disulfide bonds. This provides unique information about protein stability and folding that is not obtainable directly with other interactions, such as hydrogen bonds or van der Waals interactions (, , , ). It also makes it possible to trap in a stable form the intrinsically unstable partly-folded intermediates that define pathways of protein folding and assembly. The techniques described here have been developed primarily using bovine pancreatic trypsin inhibitor (BPTI) (, , , , , , , , ), but they have also been applied to homologues of BPTI (, ), ribonuclease A (, , , ), ribonuclease T1 (), and α-lactalbumin (, , ).